Tomato receptor-like cytoplasmic kinase Fir1 interacts with a negative regulator of jasmonic acid signaling

Plant cells detect potential pathogens through plasma membrane-localized pattern recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) and activate pattern-triggered immunity (PTI). PRR-mediated MAMP perception is linked to PTI signaling by receptor-like cytoplasmic kinases (RLCKs). In tomato, Flagellin-sensing 2 (Fls2)/Fls3 interacting RLCK 1 (Fir1) is involved in PTI triggered by flagellin perception. Fir1 is necessary for regulation of jasmonic acid (JA) signaling and is involved in pre-invasion immunity. We show that Fir1 physically interacts with JASMONATE-ZIM-DOMAIN PROTEIN 3 (JAZ3), a negative regulator of JA signaling. This finding suggests that Fir1 modulates JA signaling by regulating JAZ3.


Figure 1. Fir1 interacts with JAZ3 in planta and in vitro:
(A) The indicated proteins were fused to C-LUC or N-LUC and co-expressed via A. tumefaciens in N. benthamiana leaves. Empty vector (EV) or green fluorescent protein (GFP) were used as negative controls. Luciferase activity was quantified by measuring relative luminescence at 48 h after agro-infiltration. The experiment was repeated three times with similar results. Horizontal lines inside boxes represent median values. Median values are indicated by bold lines inside each box. The third and first quartiles of the value distribution are represented by the top and bottom sides of the boxes, respectively. The extreme values within 1.5 times the interquartile range are shown by lines extending from the boxes. Individual data points are shown as circles, and letters denote statistical significance as assessed by one-way ANOVA and Tukey's post-hoc test (P < 0.05). (B) Expression of the indicated proteins fused to the N-terminal half (N-LUC) of the luciferase protein in leaves of N. benthamiana plants. Proteins were detected by immunoblot analysis using anti-luciferase (α:LUC) antibodies. Ponceau S staining of Rubisco is shown as a loading control. Asterisks denote bands of the expected molecular weight. (C) GST or GST-Fir1 bound to glutathione resin was added to lysates of E. coli cells expressing His-JAZ3 or His-JAZ7, and incubated overnight. Proteins eluted from the glutathione resin were fractionated by SDS-PAGE and subjected to Western blot analysis with anti-His (α:His) antibodies or stained with Coomassie Brilliant Blue, as indicated. The experiment was repeated four times with similar results. In A, B and C, results of a representative experiment are shown.

Description
The plant immune system is equipped with plasma membrane-localized pattern recognition receptors (PRRs) that mediate early detection of potential pathogens by recognizing conserved microbe-associated molecular patterns (MAMPs) (DeFalco and Zipfel 2021). In tomato plants (Solanum lycopersicum), the PRR repertoire includes the Flagellin sensing 2 (Fls2) (Robatzek et al. 2007) and Flagellin sensing 3 (Fls3) (Hind et al. 2016) receptors, which recognize the bacterial flagellinderived peptides flg22 and flgII-28, respectively. Binding of flg22 and flgII-28 by Fls2 and Fls3 triggers signaling pathways that initiate a battery of immune responses, known as pattern-triggered immunity (PTI) (Liang and Zhou 2018). The output of PTI often consists of generation of reactive oxygen species (ROS), gene transcription reprogramming, activation of mitogenactivated protein kinases (MAPKs), closure of stomata, and the accumulation of antimicrobial proteins (DeFalco and Zipfel 2021).
We recently investigated the role of the tomato Fls2/3 interacting RLCK 1 (Fir1) in PTI (Sobol et al. 2022). Fir1 was found to interact with Fls2 and Fls3, suggesting that it plays a role as an early signaling component of PTI activated by flagellin perception. Phenotypic analyses revealed that a loss-of-function mutation of the fir1 gene causes increased susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, Fir1 appears to participate in pre-invasion immunity and stomatal closure. Consistent with its interaction with Fls2 and Fls3, Fir1 is required for flagellin-induced ROS production and PATHOGENESIS-RELATED 1b mRNA expression. Analysis of gene expression profiles of fir1 mutant plants upon PTI activation revealed a deregulated JA response. In line with this finding, fir1 loss-of-function mutations diminished the contribution of coronatine to virulence of Pst bacteria, and enhanced resistance to Botrytis cinerea, a necrotrophic fungus, after activation of PTI. These findings suggest that Fir1 is necessary for the appropriate activation of PTI and support a model in which Fir1 regulates JA signaling to inhibit a JA response (Thaler et al. 2012).
We hypothesized that Fir1 modulates the JA response by interacting with components of the JA signaling pathway. To assess this hypothesis, a split-luciferase complementation assay was employed to test the interaction between Fir1 and members of the JASMONATE-ZIM-DOMAIN (JAZ) protein family, which negatively regulate JA signaling (Pauwels and Goossens 2011), and whose transcripts were differentially expressed in fir1-1 mutant plants during activation of PTI (JAZ2, JAZ3, JAZ7, and JAZ9) (Sobol et al. 2022). In addition, we tested the interaction of Fir1 and the JA receptor JASMONIC ACID-INSENSITIVE1 (JAI1) (Li et al. 2004), and FER1 (homolog of Arabidopsis FERONIA), which inhibits coronatine-induced signaling to promote disease resistance (Guo et al. 2018). In these experiments, Fir1 was fused to the C-terminal fragment of the luciferase protein (C-LUC) and co-expressed via Agrobacterium tumefaciens in Nicotiana benthamiana leaves with a candidate interactor fused to the N-terminal fragment of luciferase (N-LUC). As negative controls, C-LUC-Fir1 was coexpressed with either an empty N-LUC vector or with N-LUC fused to the green fluorescent protein (GFP). The luminescence emitted from leaf discs was measured 48 hours after agro-infiltration and used to quantify interactions. Leaf disks coexpressing Fir1 and JAZ3, but not any of the other protein combinations, emitted a significantly higher luminescence than the negative controls ( Figure 1A). Western blot analysis was used to validate that each fusion protein was expressed in the inoculated plant tissues ( Figure 1B and Roberts et al. 2019).
We used an in vitro pull-down assay to confirm the interaction between Fir1 and JAZ3. Fir1 fused to the glutathione-Stransferase (GST) protein was expressed in E. coli and purified. Lysates of bacterial cells expressing His-tagged JAZ3 and JAZ7 (negative control) were incubated with immobilized GST-Fir1 or GST (negative control) and washed. Western blot analysis with anti-His antibodies detected His-JAZ3, but not His-JAZ7 among the proteins pulled-down by GST-Fir1 ( Figure  1C), suggesting a direct interaction between Fir1 and JAZ3.
JAZ3 is a negative regulator of JA signaling (Pauwels and Goossens 2011) and it has been shown to be targeted by pathogen effector proteins (Yang et al. 2017;Anderson et al. 2019). For example, the Pst type III secreted effector HopBB1 interacts with the TCP14 transcription factor, a repressor of a subset of JA responses. To activate JA response genes, and consequently increase pathogen virulence, HopBB1 reduces TCP14 abundance by coupling it with JAZ3, which sends it to degradation in the SCF COI degradation complex (Yang et al. 2017). The HaRxL10 effector protein from the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) targets JAZ3 to decrease its abundance. The reduction in JAZ3 abundance activates JA responses, and suppresses SA responses, which normally restrict Hpa growth (Anderson et al. 2019). Together, the results indicate that Fir1 interacts in planta and in vitro with JAZ3, which represents a candidate signaling component regulated by Fir1.

Bacterial strains, plant materials, and growth conditions
Nicotiana benthamiana plants (Goodin et al. 2008) were cultivated at 25°C in a phytochamber under long-day conditions (16 h/8 h, light/dark). The bacterial strains used were: Escherichia coli DH5a (Invitrogen), E. coli Rosetta (Novagen), and Agrobacterium tumefaciens GV2260 (Deblaere et al. 1985). E. coli was grown in Lysogeny broth (LB) medium at 37°C and A. tumefaciens in LB medium at 30°C with the required antibiotics.

In vitro pull-down assay
Tomato cDNA was used to PCR-amplify genes encoding JAZ3 and JAZ7 using primers 15-16 (JAZ3), or 17-18 (JAZ7) ( Table  1). JAZ3 and JAZ7 were cloned downstream and in frame to a 10xHis-tag in the pET-16b vector (Novagen). The pGEX-4T1 vector carrying Fir1 fused to GST (GST-Fir1) was prepared as described by Sobol et al. (2022). Plasmids carrying His-JAZ3, His-JAZ7, GST, and GST-Fir1 were transformed into the Rosetta E. coli strain and in vitro pull-down assays were carried out as described (Sobol et al. 2022).

Catalog number Company
Anti-Luciferase antibody produced in rabbit L0159 Sigma-Aldrich D-Luciferin sodium salt L6882 Sigma-Aldrich Monoclonal Anti-polyHistidine antibody produced in mouse H1029 Sigma-Aldrich Glutathione resin L00206 GenScript